这项工作得到了诺德基金会和国立神经疾病和中风研究所 (M.N.) 的支持。a.l.r. x 和 S. R 是博士后奖学金的接受者和 Lundbeck 基金会的博士学位。

所有程序均按照欧洲 2010/63/欧盟关于动物研究的指令进行, 并经丹麦环境和食品部 (2015-15-0201-00535) 的动物实验委员会批准。
1. 插管程序
<ol><li>套管准备 注意: 避免用无菌手套接触套管。<ol>
<li>用针架切断30G 牙针的斜面金属尖端。</li>
		<li>使用针架, 将斜面金属尖端 (大约0.3 厘米) 插入30厘米长的 PE10 油管 (聚乙烯油管 0.024 "OD x 0.011" ID), 填充 aCSF (126 毫米氯化钠, 2.5 毫米氯化钾, 1.25 毫米 NaH2PO4, 2 毫米 MgSO4, 2 毫米 CaCl2, 10 毫米葡萄糖, 26 毫米 NaHCO3;pH 值为 7.4, 当使用 95% O2和 5% CO2进行毒气时。</li>
		<li>使用装有30G 针的1毫升注射器 (30 克 x ½ "0.3 x 12") 冲洗套管与 aCSF。</li>
	</ol>
</li>
	<li>
手术 注意: 慢性厘米插管允许动物从手术过程中恢复。CM 注射是在套管植入后的第二天进行的, 重要的是可以在麻醉或醒着的动物中进行。由于这是一个康复手术, 程序应在无菌条件下进行。<ol>
<li>称鼠标 (C57BL/6JRj, 两性, 8 周), 并麻醉氯胺酮和甲苯噻嗪 (100 毫克/千克; 10 毫克/千克) 的混合物, 通过腹腔 (ip) 注射。如有必要, 在手术过程中建立小鼠半剂量氯胺酮 (50 毫克/千克)。或者, 对于慢性插管, 麻醉小鼠, 将其置于异氟醚诱导腔中 2.5-3% 异氟醚, 在大约1升/分 O2。在这种情况下, 使用鼻锥维持异氟醚麻醉在 1.5-2% 整个手术。</li>
		<li>当脚趾捏的反应停止, 呼吸变得缓慢和稳定, 把动物放在一个立体定向的框架上的加热垫。</li>
		<li>应用眼科软膏。必要时重复手术。</li>
		<li>剃掉老鼠的头部和颈部, 去掉毛皮, 用酒精拭子先消毒暴露的皮肤, 然后两次用洗必泰 (0.5%) 或碘溶液 (2%)。重复灭菌两次。 注意: 在剃须后, 改变手术悬垂以清除杂物和毛发。然后将动物披上, 保护不育的田地。</li>
		<li>对于慢性插管, 在切口部位分别管理 0.5-1 毫升利多卡因 (1 毫克/毫升和0.25 毫克/毫升) 皮下 (南卡罗来纳州)。管理丁丙诺啡 (0.05 毫克/千克) 用于术后镇痛。</li>
		<li>将鼠标固定在立体定向的框架中。在确保固定后, 无论是 intraural 或颧骨拱, 稍微倾斜头部, 使其形成一个角度的120°的身体 (图 1E)。</li>
		<li>发现头骨的一部分, 立即突出的颈部肌肉-枕嵴。用一双镊子抬起上面的皮肤, 在中线处切开大约1厘米的杏仁状的皮肤。使用棉签或眼矛控制任何结果出血。</li>
		<li>以枕嵴为参考点, 将浅层结缔组织拉出, 露出颈部肌肉。</li>
		<li>小心地将钳位放在中间的切口部位, 在前-后轴处, 将其分开。每只手上都有一对弯曲的镊子, 在头骨底部的中间加入尖端, 把肌肉拉到一边。 注意: 这应该暴露 CM, 它出现作为一个微小的倒置的三角, 概述由小脑上面和髓质下面, 在半透明的硬脑膜膜 (图 1B 和 1C) 之后。</li>
		<li>使用手术眼矛或棉签, 擦拭覆盖 CM 的硬脑膜膜。</li>
	</ol>
</li>
	<li>
插入套管入 CM
	<ol>
<li>从 aCSF 的注射器中取出套管, 使30G 针附着在油管的后端。</li>
		<li>将30G 针连接到注射器泵上的蒸馏水填充100µL 注射器上。</li>
		<li>用注射器泵取出空气, 在套管中插入大约1厘米的气泡。</li>
		<li>使用注射器泵, 提取 12 ul 的预期脑脊液示踪剂进入套管。</li>
		<li>抓住套管, 用脑脊液示踪剂, 靠近管盖针, 用一双弯曲的镊子握在支配手上。将非显性手的中指放在对面的耳条上, 并保持其稳定, 供以后用作套管的休息用。</li>
		<li>将套管插入与鼠标头相对的45°角, 通过硬脑膜所看到的三角形的角度, 将其传递到 CM 的中心。避免小脑或髓质的任何穿透。确保针只插入到深度为 1-2 毫米,即到斜角完全位于硬脑膜下的点。松开镊子持有套管, 并让套管休息的非显性的手。 注: 牙针的斜面端将需要使用一些力量刺穿硬脑膜膜覆盖 CM。</li>
		<li>如有必要, 使用手术眼矛或棉签拭去任何脑脊液渗漏。</li>
		<li>将 2-3 滴氰胶水放在套管周围的硬脑膜膜上。添加一滴胶水加速器, 立即固化胶水。用牙科水泥 (大约0.5 毫克) 和氰胶 (3-5 滴) 的混合物盖住头骨和针头。紧接着, 应用一滴胶水加速器来治疗。</li>
		<li>对于慢性插管, 切油管 (留约 2-3 厘米连接到套管), 并密封它与外科焊接保持颅内压 (ICP) 水平, 通过防止脑脊液泄漏通过油管。</li>
		<li>用于慢性插管, 管理卡洛芬 (5 毫克/千克;</li>
		<li>对于慢性插管, 把鼠标放在笼子里, 把它放在加热垫上保持体温, 直到动物完全从麻醉中恢复。 注意: 动物应该单独存放在笼子里, 以确保套管完好无损。通过将鼠标放在纸巾或其他固体基板上, 确保鼻子清晰的床上用品。 注: 第二天, 当动物从手术过程中恢复为套管插入, 他们可以注射脑脊液示踪剂。在麻醉下注射, 管理氯胺酮/甲苯噻嗪的混合物 (100 毫克/千克; 10 毫克/千克, 分别; ip), 然后继续执行2 节中介绍的步骤。对于唤醒动物的注射, 请继续执行3节。</li>
	</ol>
</li>
</ol>2. 麻醉动物急性植入 CM 导管注射脑脊液示踪剂
注: 在麻醉动物的急性植入性 CM 套管注射脑脊液示踪剂后, 在上一节步骤8后立即进行脑脊液示踪剂注射, 如下所述。
<ol><li>使用注射器泵, 以 1 ul/分5或10分钟的速度开始注射到脑脊液示踪剂的 CM 中, 从而分别产生5µL 或10µL 的总体积。在注射结束时, 允许脑脊液追踪器在整个大脑中循环30分钟, 不受干扰的套管。</li>
	<li>30分钟后, 切断连接到套管的油管 (大约4厘米的距离从针尖) 和密封它的末端使用外科焊接。</li>
	<li>在深度麻醉下, 用斩首弄死动物。快速解剖大脑和修复组织通过浸泡在4% 多聚甲醛 (粉煤灰) 稀释磷酸盐缓冲盐水 (PBS; 0.01M; pH 值 7.4) 隔夜 (o/n) 在4摄氏度。</li>
</ol>3. 通过长期植入的 CM 套管在醒动物体内注射脑脊液示踪剂
<ol><li>使用注射器泵, 提取7µL 或12µL 所需的脑脊液示踪剂组成的套管约30厘米的 PE10 导管与斜角牙科针尖0.5 厘米。</li>
	<li>轻轻地抑制动物和切割约1厘米的油管附加到套管。</li>
	<li>仍然在温和的限制下, 快速连接的套管填充脑脊液示踪剂到 CM 植入套管。</li>
	<li>使用注射器泵, 开始注射到脑脊液示踪剂的 CM 的速度为1µL/分钟. 注射7µL 或12µL, 以达到最终容积的脑脊液注射示踪5µL 或10µL, 从而弥补 aCSF 留在植入套管。在注射结束时, 允许脑脊液追踪器在整个大脑中循环30分钟, 不受干扰的套管。确保油管在注射和循环期间保持连接。</li>
	<li>脑脊液示踪循环结束时, 通过斩首进行安乐死, 保证动物被深深麻醉。快速解剖大脑和修复组织通过浸泡在4% 多聚甲醛 (粉煤灰) 稀释磷酸盐缓冲盐水 (PBS; 0.01M; pH 值 7.4) 隔夜 (o/n) 在4摄氏度。</li>
</ol>

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作者没有什么可透露的。

我们已经提出了一个协议, 描述了一个详细的程序, 一池的麦格纳插管 (CMc) 提供了一个简单的方法, 提供标签的分子到脑脊液室。CMc 允许随后的脑脊液动力学可视化, 无论是在体内和前体,使用不同的成像方式或组织学。
CMc 技术的主要优点之一在于它可以直接进入蛛网膜下腔, 而无需通过开颅手术暴露大脑。通过不需要颅骨窗口或大脑实质的穿透针尖, CMc 允许分子...

脑脊液 (CSF) 将中枢神经系统 (CNS) 沐浴在整个心室系统和蛛网膜下腔内, 这是一个解剖上定义的空间在连续体与脑室, 围绕大脑和脊髓。脑脊液的主要功能之一是提供从脑实质中清除代谢物和溶质的途径。通过最近发现的 glymphatic 系统1, 将大脑模拟到外围淋巴系统, 方便了清除。在这里, 我们描述和讨论的是一个微创的方法, 以直接提供分子进入脑脊液池的麦格纳插管 (CMc)。CMc 是研究 glymphatic 功能的关键方法。此外, CMc 也可以应用于脑脊液动力学研究和快速, 大脑范围内的非血脑屏障 (BBB) 渗透分子进入脑实质, 沿周围空间。
CMc 通过中枢神经系统利用脑脊液运动动力学的生理原理, 将标记的示踪分子或药物传送到麦格纳池 (CM) 的脑脊液填充空间。分子通过插入到寰枕硬脑膜膜内的套管注入, 覆盖厘米. 然后通过 paravascular 空间1, 将脑脊液散装流进入脑实质。通过 CMc 注射的示踪剂或造影药剂跟随脑脊液的运动, 它允许评估脑脊液运动和 glymphatic 的流入, 量化的强度水平的标记分子进入大脑实质。CMc 兼容不同的成像技术, 包括荧光, 2 光子显微术, 磁共振成像 (MRI)。此外, 可以在体内或前体中执行此评估。重要的是, CMc 允许 glymphatic 系统的可视化在麻醉下或在自然睡眠期间, 并且在醒的, 自由地移动的动物。
CMc 技术可用于研究脑脊液中流体动力学的不同方面, 但已证明对研究 glymphatic 系统特别有用。Glymphatic 活动通过 aquaporin-4 (AQP-4) 水通道在胶质血管包裹 endfeet 的膜中拴在 periarterial 空间中, 推动脑脊液的对流流动。对流流使脑脊液和间质流体在脑实质中相互交换。通过 perivenous 空间2,3, 将含有代谢废物和溶质的 CSF 从脑实质中移除。最终, CSF 通过最近描述的硬脑膜淋巴管到达外围,4,5。对于清除有害废物代谢物 (如淀粉样蛋白β2), glymphatic 系统已被证明是至关重要的。进一步, glymphatic 清除在老化6, 在创伤性脑损伤以后7, 并且在糖尿病的动物模型8和阿尔茨海默病9。值得注意的是, glymphatic 活动是状态依赖性的, 在睡眠或麻醉中显示的活动显著高于清醒的1。事实上, 年轻的麻醉动物表现出最高的 glymphatic 活动。因此, 在研究其在健康和疾病中的作用时, glymphatic 活动的实验量化是至关重要的。
几项研究已经讨论了脑脊液动力学及其与间质流体的交换在脑实质。然而, 标记的分子被提供的方法是相当侵入性的, 触发脑实质损伤和颅内压的变化 (ICP) (见回顾10)。一些例子是脑室内或脑实质注射, 涉及开颅手术或钻孔在头骨的一个毛刺孔。这些过程已被证明可以改变 ICP, 从而中断 glymphatic 函数2。此外, 这种侵入性方法诱发 astrogliosis 和增加 AQP-4 免疫反应在脑实质受损地区及其周围的11,12。由于星形胶质细胞和 AQP-4 是 glymphatic 系统的关键元素, CMc 是其研究的选择方法。CMc 与更多侵入性程序相比, 其主要优点是保持完整的颅骨和脑实质, 避免 ICP 改变和 astrogliosis。因此, CMc 与不同的成像工具一起打开了广泛的可能性, 不仅研究 glymphatic 系统, 而且还有流动的动力学和机制的动态平衡, 以及动物模型的神经系统疾病。
该池 (CMc) 程序允许容易和直接进入脑脊液 (CSF)。通过注射不同的分子 (例如荧光示踪剂, MRI 造影药), 实验者可以跟踪他们的运动在脑脊液室和评估 glymphatic 系统的活动。下面的协议描述了急性 CMc, 在手术后立即注射, 和慢性套管植入, 其中动物从手术过程中恢复为以后注射。急性和慢性植入的最重要的区别是慢性植入允许研究醒鼠的 glymphatic 活动。

<table border="0" cellpadding="0" cellspacing="0" width="1153">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">SOPIRA Carpule 30G 0.3 x 12mm</td>
			<td style="width:245px;">Kulzer</td>
			<td style="width:257px;">AA001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Polyethylene Tubing 0.024&#8221; OD x 0.011&#8221; ID</td>
			<td style="width:245px;">Scandidact</td>
			<td>PE10-CL-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30G x &#189;&#8221; 0.3 x 12 mm Luer-Lock</td>
			<td style="width:245px;">Chirana T. Injecta</td>
			<td>CHINS01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:485px;">Chlorhexidine 0.5% (chlorhexidine digluconate)</td>
			<td style="width:245px;">Meda AS</td>
			<td style="width:257px;">no catalogue number, see link in comments</td>
			<td>http://www.meda.dk/behandlingsomrader/desinfektion/desinfektion-af-hud/klorhexidin-sprit-medic-05/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alcohol Swab 70% Isopropyl Alcohol 30 x 60mm</td>
			<td>Vitrex Medical A/S</td>
			<td>520213</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Viskoese Oejendraeber Ophtha</td>
			<td>Ophtha</td>
			<td>145250</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:485px;">Wooden applicator, Double cotton bud (&#216; appr. 4 - 5 mm, length appr. 12 mm)</td>
			<td style="width:245px;">Heinz Herenz</td>
			<td style="width:257px;">1032018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eye spears</td>
			<td>Medicom</td>
			<td>A18005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ferric chloride 10% solution</td>
			<td style="width:245px;">Algeos</td>
			<td style="width:257px;">NV0382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kimtech Science Precision Wipes Tissue Wipers</td>
			<td style="width:245px;">Kimberly Clark Professional</td>
			<td style="width:257px;">05511</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Loctite Super Glue Precision 5g</td>
			<td style="width:245px;">Loctite</td>
			<td style="width:257px;">no catalogue number, see link in comments</td>
			<td>http://www.loctite-consumer.dk/da/produkter/superglue-liquid.html&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insta-Set CA Accelerator</td>
			<td style="width:245px;">Bob Smith Industries</td>
			<td style="width:257px;">BSI-152</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dental Cement Powder</td>
			<td style="width:245px;">A-M Systems</td>
			<td style="width:257px;">525000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical weld&#160;</td>
			<td style="width:245px;">Kent Scientific Corporation</td>
			<td style="width:257px;">INS750391</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:485px;">Hamilton syringe GASTIGHT&#174; , 1700 series, 1710TLL, volume 100 &#956;L, PTFE Luer lock</td>
			<td>Hamilton syringes</td>
			<td style="width:257px;">1710TLL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">LEGATO 130 Syringe pump</td>
			<td style="width:245px;">KD Scientific</td>
			<td style="width:257px;">788130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Paraformaldehyde powder, 95%</td>
			<td style="width:245px;">Sigma Aldrich</td>
			<td style="width:257px;">158127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Phosphate buffered saline (PBS; 0.01M; pH 7.4)</td>
			<td style="width:245px;">Sigma Aldrich</td>
			<td style="width:257px;">P3813</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Ovalbumin, Alexa Fluor 647 Conjugate</td>
			<td style="width:245px;">Thermo Fisher Scientific</td>
			<td style="width:257px;">O34784</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:485px;"> 
			DAPI (diamidino-2-phenylindole) Solution (1 mg/mL)</td>
			<td style="width:245px;">Thermo Fisher Scientific</td>
			<td style="width:257px;">62248</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Dextran, Fluorescein, 3000 MW, Anionic</td>
			<td style="width:245px;">Thermo Fisher Scientific</td>
			<td style="width:257px;">D3305</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E-Z Anesthesia EZ-7000 Classic System</td>
			<td style="width:245px;">E-Z Systems</td>
			<td>EZ-7000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Attane Isofluran 1000 mg/g</td>
			<td style="width:245px;">ScanVet</td>
			<td>55226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Euthanimal 200mg/mL (sodium pentobarbital)</td>
			<td style="width:245px;">ScanVet</td>
			<td style="width:257px;">545349</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Ketaminol Vet 100 mg/mL (ketamine)</td>
			<td>Intervet International BV</td>
			<td style="width:257px;">511519</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:485px;">Rompin Vet 20 mg/mL (xylazin)</td>
			<td style="width:245px;">KVP Pharma + Veterin&#228;r Produkte GmbH</td>
			<td style="width:257px;">148999</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Xylocain 20 mg/mL (lidocain)</td>
			<td style="width:245px;">AstraZeneca</td>
			<td style="width:257px;">158543</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Marcain 2.5 mg/mL (bupivacain)</td>
			<td style="width:245px;">AstraZeneca</td>
			<td style="width:257px;">123918</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:485px;">Bupaq Vet 0.3 mg/mL (buprenorphine)</td>
			<td style="width:245px;">Richter Pharma AG</td>
			<td style="width:257px;">185159&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

cannula implantation into the cisterna magna of rodents

大池插管 (CMc) 是一个简单的程序, 使直接进入脑脊液 (CSF) 没有手术损伤的头骨或脑实质。在麻醉啮齿目动物中, 硬脑膜通过颈部肌肉的钝夹层暴露, 使套管插入到麦格纳 (CM) 的池中。套管由细斜面针或硼硅酸盐毛细管组成, 通过聚乙烯 (PE) 管连接到注射器。使用注射器泵, 分子可以直接注射在控制速率的 CM, 这是连续的蛛网膜下腔。从蛛网膜下腔, 我们可以通过对流流的周围空间来追踪脑脊液的通量, 从而产生与间质流体的溶质交换。CMc 可在手术后立即注射, 或用于慢性植入, 随后注射麻醉或清醒, 自由移动啮齿动物。定量的示踪剂分布在脑实质可以由荧光, 2 光子显微术, 磁共振成像 (MRI), 根据物理化学性质的注射分子。因此, CMc 结合各种成像技术, 为评估 glymphatic 系统和脑脊液动力学和功能提供了有力的工具。此外, CMc 可以作为一个管道, 快速, 大脑范围内传递的信号分子和代谢基质, 否则不能通过血脑屏障 (BBB)。

在立体定向的框架内固定小鼠或大鼠时, 枕顶区域周围的颈部肌肉被直接解剖, 以暴露出麦格纳 (厘米)。在小脑的尾端和髓质 (图 1A-1C) 之间, CM 的三角形结构容易被识别。套管通过轻轻刺穿寰枕膜 (图 1D), 插入到 CM 中 1-2 毫米。硬脑膜膜是一个强硬的结构和插入的套管是改善通过倾斜的动物头部的120°有关的身体。在...

Watch this Scientific Journal Video about Cannula Implantation into the Cisterna Magna of Rodents at JoVE.com

Cannula Implantation into the Cisterna Magna of Rodents

将套管植入啮齿动物的麦格纳池中

在这里, 我们描述了一个协议, 以执行麦格纳插管 (CMc), 一种微创的方式, 提供示踪剂, 基质和信号分子进入脑脊液 (CSF)。结合不同的成像方式, CMc 使 glymphatic 系统和脑脊液动力学评估, 以及大脑范围内的各种化合物的交付。

Cisterna magna cannulation (CMc) is a straightforward procedure that enables direct access to the cerebrospinal fluid (CSF) without operative damage to ...

cannula-implantation-into-the-cisterna-magna-of-rodents

Research

JoVE Journal

Neuroscience

43.7K 次观看.  University of Copenhagen. 在这里, 我们描述了一个协议, 以执行麦格纳插管 (CMc), 一种微创的方式, 提供示踪剂, 基质和信号分子进入脑脊液 (CSF)。结合不同的成像方式, CMc 使 glymphatic 系统和脑脊液动力学评估, 以及大脑范围内的各种化合物的交付。

视频: Cannula Implantation into the Cisterna Magna of Rodents

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

隔离啮齿类动物或人类的鼻嗅干细胞

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

科研

神经科学

Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents

Partially isolated cortex ("undercut") is an animal model of posttraumatic epileptogenesis. The surgical procedure involves cutting through the sensorimotor cortex and the underneath white matter (undercut) so that a specific region of the cerebral cortex is largely isolated from the neighboring cortex and subcortical regions1-3. After a latency of two or more weeks following the surgery, epileptiform discharges can be recorded in brain slices from rodents1; and electrical or behavior seizures can be observed in vivo from other species such as cat and monkey4-6. This well established animal model is efficient to generate and mimics several important characteristics of traumatic brain injury. However, it is technically challenging attempting to make precise cortical lesions in the small rodent brain with a free hand. Based on the procedure initially established in Dr. David Prince's lab at the Stanford University1, here we present an improved technique to perform a surgery for the preparation of this model in mice and rats. We demonstrate how to make a simple surgical device and use it to gain a better control of cutting depth and angle to generate more precise and consistent results. The device is easy to make, and the procedure is quick to learn. The generation of this animal model provides an efficient system for study on the mechanisms of posttraumatic epileptogenesis.

Partially isolated cortex (&#x201C;undercut&#x201D;) is an efficient animal model of posttraumatic epileptogenesis. Here we demonstrate how to make a novel surgical device and use it to make more precise and consistent lesions to generate this model.

准备在鼠害外伤性癫痫的底切模型

Partially isolated cortex ("undercut") is an animal model of posttraumatic epileptogenesis. The surgical procedure involves cutting through the ...

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

一个多认知功能测试，并记录在啮齿动物的神经元活动的完全自动化和高度灵活的系统

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

方法以使对新生儿鼠害的腹头骨开颅手术

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

低聚淀粉样蛋白-β的立体灌注到小鼠海马

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, dystonia or tremor. DBS is based on the delivery of electrical stimuli to specific deep anatomic structures of the central nervous system. However, the mechanisms underlying the effect of DBS remain enigmatic. This has led to an interest in investigating the impact of DBS in animal models, especially in rats. As DBS is a long-term therapy, research should be focused on molecular-genetic changes of neural circuits that occur several weeks after DBS. Long-term DBS in rats is challenging because the rats move around in their cage, which causes problems in keeping in place the wire leading from the head of the animal to the stimulator. Furthermore, target structures for stimulation in the rat brain are small and therefore electrodes cannot easily be placed at the required position. Thus, a set-up for long-lasting stimulation of rats using platinum/iridium electrodes with an impedance of about 1 M&#937; was developed for this study. An electrode with these specifications allows for not only adequate stimulation but also recording of deep brain structures to identify the target area for DBS. In our set-up, an electrode with a plug for the wire was embedded in dental cement with four anchoring screws secured onto the skull. The wire from the plug to the stimulator was protected by a stainless-steel spring. A swivel was connected to the circuit to prevent the wire from becoming tangled. Overall, this stimulation set-up offers a high degree of free mobility for the rat and enables the head plug, as well as the wire connection between the plug and the stimulator, to retain long-lasting strength.

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

微电极引导电极植入到大鼠丘脑底核的长期深部脑刺激

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

背根神经节的啮齿动物的脊髓和隔离的液压挤压

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

Operant Protocols for Assessing the Cost-benefit Analysis During Reinforced Decision Making by Rodents

Reinforcement-guided decision making is the ability to choose between competing courses of action based on the relative value of the benefits and their consequences. This process is integral to the normal human behavior and has been shown to be disrupted by neurological and psychiatric disorders such as addiction, schizophrenia, and depression. Rodents have long been used to uncover the neurobiology of human cognition. To this end, several behavioral tasks have been developed; however, most are non-automated and are labor-intensive. The recent development of the open-source microcontroller has enabled researchers to automate operant-based tasks for assessing a variety of cognitive tasks, standardizing the stimulus presentation, improving the data recording and consequently, improving the research output. Here, we describe an automated delay-based reinforcement-guided decision-making task, using an operant T-maze controlled by custom-written software programs. Using these decision-making tasks, we show the changes in the local field potential activities in the anterior cingulate cortex of a rat whilst it performs a delay-based cost-and-benefit decision-making task.

A cost-benefit analysis is a weighing-scale approach that the brain performs during the course of decision making. Here, we propose a protocol to train rats on an operant-based decision-making paradigm where rats choose higher rewards at the expense of waiting for 15 s to receive them.

啮齿动物强化决策中成本效益分析的操作协议

Reinforcement-guided decision making is the ability to choose between competing courses of action based on the relative value of the benefits and their ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

拉米内切手术和脊髓窗口植入小鼠

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.